Responses of Anabaena sp. PCC7120 to lindane: Physiological effects and differential expression of potential lin genes

Abstract Lindane (γ‐HCH) is an organochlorine pesticide that causes huge environmental concerns worldwide due to its recalcitrance and toxicity. The use of the cyanobacterium Anabaena sp. PCC 7120 in aquatic lindane bioremediation has been suggested but information relative to this process is scarce. In the present work, data relative to the growth, pigment composition, photosynthetic/respiration rate, and oxidative stress response of Anabaena sp. PCC 7120 in the presence of lindane at its solubility limit in water are shown. In addition, lindane degradation experiments revealed almost a total disappearance of lindane in the supernatants of Anabaena sp. PCC 7120 culture after 6 days of incubation. The diminishing in lindane concentration was in concordance with an increase in the levels of trichlorobenzene inside the cells. Furthermore, to identify potential orthologs of the linA, linB, linC, linD, linE, and linR genes from Sphingomonas paucimobilis B90A in Anabaena sp. PCC 7120, a whole genome screening was performed allowing the identification of five putative lin orthologs (all1353 and all0193 putative orthologs of linB, all3836 putative orthologs of linC, and all0352 and alr0353 putative orthologs of linE and linR, respectively) which could be involved in the lindane degradation pathway. Differential expression analysis of these genes in the presence of lindane revealed strong upregulation of one of the potential lin genes of Anabaena sp. PCC 7120.

diversity, organization, and distribution of lindane catabolic pathways are reasonably well-established in these bacteria (Verma et al., 2014). Aerobic lindane degradation pathways are conformed by lin genes and are subdivided into upper and lower pathways. In particular, the lin pathways are well-characterized in Sphingobium japonicum UT26S (Nagata et al., 1999(Nagata et al., , 2007 and Sphingobium indicum B90A (Lal et al., 2010).
PCC 7120, a nitrogen-fixing cyanobacterium, was reported to be able to metabolize lindane (γ-HCH) yielding pentachlorocyclohexene and 1,2,4trichlorobenzene in a process dependent on nir operon, which encodes some enzymes for nitrate utilization (Kuritz & Wolk, 1995;Kuritz et al., 1997). However, the genes and subsequent catabolic pathways involved in such a process remain unknown. In addition, the effect of the presence of lindane on the photosynthetic apparatus of Anabaena sp.
PCC 7120 was analyzed, concluding that photosynthetic activity was slightly increased in the presence of lindane and that no changes were observed in the synthesis and activity of ferredoxin-NADP + reductase (FNR) (Bueno et al., 2004).
On the other hand, it was previously reported that the levels of lindane in supernatants of M. aeruginosa PCC 7806 cultures treated with 7 mg/L lindane decreased significantly after 15 days of treatment (Ceballos-Laita et al., 2015). Nevertheless, only an ortholog of the linC gene was found in the genome of M. aeruginosa NIES-843, which interestingly was induced in the presence of lindane (Sarasa-Buisán et al., 2022).
The lin pathway probably constitutes a unique biochemical system that allows HCH detoxification. Until the moment the genes that constitute this pathway and the substrate specificity, isomer coverage, and kinetics of enzymes are relatively well-known in Sphingomonads. However, information concerning this pathway in other organisms is scarce (Lal et al., 2010). The knowledge of the presence of lin genes as well as the tolerance of Anabaena to lindane is important to evaluate the potential use of this model cyanobacterium in bioremediation strategies or in other processes such as the development of whole-cell biosensors to detect HCH isomers.
In this work, Anabaena sp. PCC 7120 physiological responses to the presence of lindane, namely growth rate, pigment composition, photosynthetic/respiration rates, and oxidative stress response were evaluated.
Moreover, a search of lin genes in the genome of this cyanobacterium was performed and their transcriptional responses in the presence of lindane were studied. Finally, Anabaena sp. PCC 7120 degradative capacity was also studied by testing the disappearance of lindane in the supernatants of cells treated with this pesticide as well as the appearance of lindane metabolic intermediaries inside Anabaena sp. PCC 7120 cells.

| Growth measurement
The effect of 7 mg/L of γ-HCH on the photoautotrophic growth of three independent cultures of Anabaena sp. PCC 7120 with an initial OD 750nm of 0.3 was measured spectrophotometrically using a Cary 100 Bio UV-Visible spectrophotometer (Varian). The optical density was recorded at 750 nm for 22 days every 48 h. At the same time, the packed cell volume (PCV) was measured using 5 mL graduated tubes with a capacity of 60 μL of packed volume. Five milliliters of cultures were centrifuged in the aforementioned tubes for 5 min at 18°C and 2000xg using an Allegra X 30 R centrifuge. PCV measurements were expressed as microliters of PCV per milliliter of culture.

| Pigment content determinations
Pigment content of Anabaena sp. PCC 7120 in the presence of 7 mg/L of γ-HCH was determined at exponential (OD 750nm = 1.0) and stationary (OD 750nm = 2.0) phases of growth in three independent cultures of each condition with an initial OD 750nm of 0.3. Chl a, phycobiliprotein, and carotenoid levels were quantified as described by Mackinney (1941), Glazer (1976), and Davies (1976), respectively.

| Net photosynthesis and dark respiration rate measurements
Net photosynthesis and dark respiration rates of Anabaena sp. PCC 7120 were measured at room temperature with a Clark-type oxygen electrode model Chlorolab 2 (Hansatech). Net photosynthesis (defined as true photosynthesis minus photorespiration and dark respiration (Wohlfahrt & Gu, 2015), was determined by measuring the O 2 increase during 5 min illuminating cell suspensions with white light at 10, 50, 100, and 400 µmol photons·m −2 ·s −1 . Dark respiration was determined by measuring the consumption of oxygen during 3 min in darkness. Both rates were measured at exponential (OD 750nm = 1.0) and stationary (OD 750nm = 2.0) phases of growth and were expressed as pmol O 2 × s −1 /μL of PCV. All measurements were performed in triplicate.

| Catalase and superoxide dismutase (SOD) activity determinations
Catalase and SOD activities were measured after 48 h of exposure to 7 mg/L of lindane in three independent cultures with an initial OD 750nm of 0.3. 50 mL of cell culture were centrifuged at 3000 g and 4°C for 10 min and cells were resuspended in 1 mL of phosphate buffer (KH 2 PO 4 /K 2 HPO 4 ) 50 mM pH 7. Then cells were broken by sonication, the resulting solution was centrifuged at 13,000 g and 4°C for 10 min and the total protein concentration was determined in the supernatant by using the BCA™ Protein Assay kit (Thermo Fisher Scientific).
Catalase activity was determined as described by Beers and Sizer (1952). Six hundred micrograms of protein extract was mixed with H 2 O 2 to a final concentration of 20 mM and the dissociation of H 2 O 2 was followed at 240 nm with a Cary 100 Bio UV-Visible spectrophotometer (Varian) for 5 min. Catalase activity was expressed in units per milligram of total protein, defining a unit as the amount of enzyme that dissociates 1 μg of H 2 O 2 per minute.
SOD activity was measured as described by Winterbourn et al. (1975) with some modifications implemented by Sein-Echaluce et al. (2015). Six hundred micrograms of protein extract was mixed with 6.4 mM ethylenediaminetetraacetic acid (EDTA), 41 μM nitro-blue tetrazolium (NBT), 2.3 μM riboflavin and 23.5 μM N,N,N′,N′-Tetramethylethylenediamine (TEMED) and SOD activity was determined by measuring the inhibition of NBT reduction by SOD. Thus, absorbance at 560 nm was determined before and after illuminating the mixtures for 10 min with UV light, using a control containing phosphate buffer instead of the protein extract. SOD activity was expressed in units per milligram of total protein, defining a unit as the amount of enzyme that inhibits the maximum reduction to half.

| Microscopy experiments
To analyze the effects of lindane on the morphology of Anabaena sp.
PCC 7120 filaments, bright-field, and fluorescence microscopic examinations were carried out with a Nikon Eclipse 50i Epifluorescence microscope. Photographs were taken with a Nikon DXM1200F camera coupled to the microscope both at exponential (OD 750nm = 1.0) and stationary (OD 750nm = 2.0) phases of growth.
Fluorescence was detected using a 560/40 nm excitation filter, a 595 nm dichroic beam splitter, and a 630/60 nm emission filter.
2.7 | Identification of putative lin genes in the genome of Anabaena sp. PCC 7120 To identify putative lin genes in the genome of Anabaena sp. PCC 7120 the sequences of the proteins encoded by genes linA, linB, linC, linD, linE, and linR from S. paucimobilis B90A were retrieved from Uniprot (https://www.uniprot.org/). Using these sequences as queries and an expectancy-value threshold of 0.005, a protein BLAST (Basic Local Alignment Search Tool) using the CyanoBase Similarity Search (http://genome.microbedb.jp/blast/blast_search/cyanobase/ genes) was performed on Anabaena sp. PCC 7120 genome. In all cases, the sequence with the lowest expected value was selected and a pairwise global alignment using ClustalW (https://www.genome.jp/ tools-bin/clustalw) was performed using default settings.

| Degradation measurements
The degradation of lindane was studied in three independent cultures with an initial OD 750nm of 0.6 containing 7 mg/L of lindane.
Erlenmeyer flasks containing 7 mg/L of lindane in BG-11 medium without cells served as controls for evaporation and photodegradation. After 1, 3, and 6 days of treatment, 10 mL of each sample was taken and centrifuged for 5 min at 3000 g in the case of Anabaena sp.
PCC 7120 cultures. Lindane analysis of these samples was carried out in the HCH Analysis laboratories at the facilities of the new security cell in Bailín II (Sabiñánigo, Huesca). The isomers of HCH in an aqueous matrix were extracted using a liquid-liquid extraction with hexane and the quantification of the remaining amount of each isomer was carried out using gas chromatography with a mass detector (Agilent Series 7890 A). The identification of isomers was carried out using commercial individual standards.
The formation of degradation intermediaries was studied in three independent cultures with an initial OD 750nm of 0.6 containing 7 mg/L of lindane. Ten milliliters of cultures were taken after 0 (control), 24, and 48 h of exposure to lindane and then centrifuged for 5 min at 3000 g. A pellet of cells was extracted by adding 2 mL of hexane at 4°C and shaking in a vortex for 5 min. Finally, the resulting suspension was centrifuged at 12,000 g and 4°C for 10 min and the supernatant was used to determine the presence of lindane degradation intermediaries.
One microliter of diluted samples were analyzed in a gas chromatography coupled to a mass detector using a 436GC with an initial OD 750nm of 0.3. RNA was extracted from 25 mL of each culture after 12 and 24 h of exposure to 7 mg/L of lindane following a method described in Sarasa-Buisan et al.
(2022). The absence of DNA in the RNA samples was checked by real-time PCR, using oligonucleotides for the housekeeping gene rnpB (Vioque, 1992). RNA was quantified spectrophotometrically using a SPECORD ® PLUS Analytik Jena spectrophotometer. , and carotenoids (c) in Anabaena sp. PCC 7120 cells exposed to 7 mg/L of lindane was determined at exponential (OD 750nm = 1.0) and stationary (OD 750nm = 2.0) growth phases. Since DMSO was used to dissolve lindane in the preparation of stock solutions, a control culture in which the same amount of DMSO alone was added to cells is also included. Three biological replicates were used for each sample. Values were normalized to packed cell volume and expressed as micrograms of pigment × μL −1 packed cell volume. The average of the three measurements is represented and the standard deviation is included. ns not significant, **p < 0.01, obtained with a t-test analysis comparing each data with respect to its control.
Two micrograms of total RNA was reverse-transcribed using SuperScript retrotranscriptase (Invitrogen) following the manufacturer's conditions. Real-time PCR was performed using the ViiA™ 7 real-time PCR System (Applied Biosystems). The specific primers for each gene that were analyzed are included in Appendix: Table A1. Each reaction was set up by mixing 12.5 µL of SYBR Green PCR Master Mix with 0.4 μL of 25 µM primer mixture and 10 ng of cDNA template in a final volume of 30 µL.
The extension of PCR products was performed at 60°C. The relative mRNA levels of the target genes were normalized to the housekeeping gene rnpB (Vioque, 1992). Relative quantification was performed according to the comparative Ct method (ΔΔCt Method) (Livak & Schmittgen, 2001). The minimum fold-change threshold was set up to ±1.5-fold.

| Statistical tools
To determine whether the changes observed in the measurements of different parameters were significant, t-test statistical analyses were performed by using the GraphPad Prism 7 program. μmol photons m -2 s -1 ) and dark respiration rate (e) were measured at exponential (OD 750nm = 1.0) and stationary (OD 750nm = 2.0) growth phases in Anabaena sp. PCC 7120 cells exposed to 7 mg/L of lindane. Since DMSO was used to dissolve lindane in the preparation of stock solutions, a control culture in which the same amount of DMSO alone was added to cells is also included. Three biological replicates were used for each sample. Values were normalized to packed cell volume and expressed as pmol O 2 s −1 × μL −1 packed cell volume. The average of the three measurements is represented and the standard deviation is included. ns, no significant, *p < 0.05, ***p < 0.001, ****p < 0.0001, obtained with a t-test analysis comparing each data with respect to its control.
corresponded to the solubility limit of lindane in water. The growth rate was measured by using the optical density at 750 nm as well as the PCV (Figure 1a,b). Since DMSO was used to solve lindane, a control culture in which DMSO alone was added to the cells was monitored. Growth curves representing both parameters, OD at 750 nm and PCV showed similar growth of Anabaena in the presence and absence of lindane (Figure 1a,b). Indeed, the doubling time was

| Lindane triggered oxidative stress in
Anabaena cells Results shown in Figure 4 indicated that the expression of sodA was slightly downregulated but the expression of cat was strongly upregulated in these conditions (four-fold after 12 h of lindane treatment and 40-fold after 24 h of lindane treatment). In addition, superoxide dismutase and catalase activities were also determined in cultures after their exposure to 7 mg/L of lindane.
Results are shown in Table 1. Catalytic activities were in agreement with transcriptional data since a significant increase in catalase activity was observed in cells exposed to lindane whereas the SOD activity was not altered in these conditions (Table 1). In summary, these results strongly suggest the induction of the oxidative stress response in Anabaena sp. PCC 7120 cells as a consequence of the presence of lindane.

| Anabaena sp. PCC 7120 cells were able to metabolize lindane
Lindane degradation experiments were performed with Anabaena PCC 7120 by using a BG11-modified media lacking ammonia to avoid inhibition of lindane metabolization (Kuritz & Wolk, 1995). Cultures

| Anabaena genome contains some putative lin genes
Orthologs of lin genes from S. paucimobilis were searched into the genome of Anabaena PCC 7120 by performing protein BLAST analyses (Table 2). In the case of linB, (haloalkane dehalogenase) two possible orthologs were found. The gene of Anabaena sp. PCC 7120 with the highest similarity was all1353 (41% similarity), which was then called linB1 (Appendix: Figure A1a). However, another ortholog (all0193) was also found. Its similarity, albeit lower, was also significant (33%) but was interestingly annotated as a haloalkane dehalogenase, and was then called linB2 (Appendix: Figure A1b).
Interestingly, a S. japonicum UT26 linC ortholog was previously PCC 7120 cells were exposed to 7 mg/L of lindane and gene expression was analyzed after 12 and 24 h of treatment. As can be seen in Figure 6   differences observed in Anabaena sp. PCC 7120 cultures treated with lindane both in exponential and stationary growth phases (Appendix: Figure A2). However, the enrichment of antenna complexes in carotenoids is a well-established process that allows the dissipation of free radicals generated by oxidative stress in cyanobacteria (Xiao et al., 2011). Thus, although cells seem not to be affected by lindane, the increase in carotenoid content joined to the strong upregulation of catalase expression suggest that lindane is triggering the oxidative stress response in Anabaena cells.
Surprisingly, we observed an increase in the photosynthetic rate in Anabaena cells treated with lindane. Although these data had been previously reported (Bueno et al., 2004), these results seem to be quite intriguing. One possible explanation could be the fact that as  (Nagata et al., 1999), at least in the conditions tested in the present work. It is important to note that Anabaena is a photoautotrophic organism and, under light conditions, this organism uses preferably photosynthetic-derived carbon sources for growth.
However, the degradation of lindane by Anabaena grown under dark conditions has never been studied. On the other hand, some bacteria uptake accidentally compounds that they would not metabolize, but if they have enzymes with a low degree of specificity for their main substrates, these compounds can be transformed by these enzymes in other products, and this could be the case of Anabaena and lindane.
Finally, the reason why the respiration rate decreased in Anabaena cells in the presence of lindane is unknown but maybe the alterations in the expression of enzymes that carry out the oxidative stress response in the darkness can be modifying the consumption of oxygen as happened in the production of oxygen by photosynthesis in the presence of lindane.
Regarding lindane metabolization, in this work, we observed that lindane content decreased in supernatants of Anabaena sp. PCC 7120 cultures. Simultaneously an increase in the presence of trichlorobenzene is observed inside Anabaena sp. PCC 7120 cells. These data are in agreement with previous results reported by Kuritz and Wolk (1995), but the difference relies on the amount of lindane added to the cultures. In this work, 7 mg/L of lindane was added to the cultures, whereas in the experiments performed by Kuritz and Wolk, assays were carried out by using 0.5 μg/L of lindane. In this work, the authors found an increase in γ-pentachlorocyclohexene and 1,2,4trichlorobenzene inside the cells treated with lindane. Since both metabolites disappeared over time, our interest was to find other metabolic intermediaries inside Anabaena cells that were similar to those of the Sphingomonas lindane degradation pathway. However, this task was unsuccessful because 2-5-dichlorophenol (resulting product of LinB enzyme), 2,5-dichlorohidroquinone (resulting product of LinC enzyme), and chlorohydroquinone (resulting product of LinD enzyme) were not detected inside Anabaena cells. Intriguingly, the protein sequence similarity searches of lin genes indicated that Anabaena contains two putative orthologs of linB (linB1 and linB2) and linC genes so that the enzymatic reactions catalyzed by these enzymes, as well as the degradation pathway after TCB remains unknown. According to our search of putative lin genes, an ortholog of linA (HCH dechlorinase) was not found in the genome of Anabaena sp. PCC 7120. In Sphingomonas two initial dechlorination reactions carried out by linA produce 1,3,4,6-tetrachloro-1,4-cyclohexadiene which presumptively is spontaneously dechlorinated to yield 1,2,4 trichlorobenzene. Since we found 1,2,4 trichlorobenzene inside Anabaena cells, another HCH dechlorinase or a similar, functionally equivalent enzyme must be catalyzing this reaction in this cyanobacterium.
In relation to Sphingomonas, although lin genes in Anabaena are just proposed, it is interesting to note the features concerning lin